Part of the work performed during the development of this invention was supported by U.S. Government funds. The U.S. Government may have certain rights in this invention.
1. Field of the Invention
This invention relates to transgenic non-human animals comprising a disrupted endothelial nitric oxide synthase gene. This invention also relates to methods of using these transgenic animals to screen compounds for activity against vascular endothelial disorders such as hypertension, stroke, and atherosclerosis, as well as for wound healing activity; methods of treating a patient suffering from a vascular endothelial disorder; methods of making the transgenic animals; and cell lines comprising a disrupted eNOS gene.
2. Related Art
In 1980, Furchgott and Zawadzzi first proposed the existence of endothelium derived relaxing factor or EDRF, later identified as nitric oxide. Furchgott (1980); Furchgott (1988); Ignarro (1988); Palmer (1987). Nitric oxide is an important messenger molecule produced by endothelial cells, neurons, macrophages, and other tissues. Marietta (1989); Moncada (1991); Nathan (1992); Snyder (1992); and Dawson et al. (1992). Since nitric oxide is a gas with no known storage mechanism, it diffuses freely across membranes and is extremely labile. Nitric oxide has a biological half-life on the order of seconds.
Nitric oxide exhibits several biochemical activities. This compound can bind to and activate soluble guanyl cyclase, resulting in increased cGMP levels. Nitric oxide also modifies a cysteine residue in glyceraldehyde-3-phosphate dehydrogenase by adenosine diphosphate ribosylation, Zhang and Snyder (1992), Katz et al. (1992), and Dimmeler et al. (1992), or S-nitrosylation via NAD interactions, McDonald and Moss (1993). Nitric oxide also binds to a variety of iron- and sulphur-containing proteins, Marletta (1993), and may have other modes of action as well.
Nitric oxide formation is catalyzed by the nitric oxide synthase enzymes (NOS). These enzymes act by producing nitric oxide from the terminal guanidino nitrogen of arginine, with the stoichiometric production of citrulline. There are several NOS isoforms encoded by separate genes. Marletta (1993), and Lowenstein and Snyder (1992). The various NOS isoforms are about 50-60% homologous overall. Some forms of NOS are found in most tissues. The different NOS isoforns: neuronal NOS (nNOS), macrophage NOS (iNOS), and endothelial NOS (eNOS), are now known as type I NOS, type II NOS and type III NOS, respectively. The properties of these NOS isoforms are summarized in the following Table:
The ubiquitous presence of blood vessels and nerves means that the endothelial and neuronal isoforms may be present in most tissues. The expression of the endothelial and neuronal isoforms can also be induced in cells that normally do not express them. The sequence of these isoforms have been published or are available in Genbank under the following accession numbers:
Each of these sequences are expressly incorporated herein by reference.
In blood vessels, the endothelial NOS isoform mediates endothelium-dependent vasodilation in response to acetylcholine, bradykinin, and other mediators. Nitric oxide also maintains basal vascular tone and regulates regional blood flow. Nitric oxide levels increase in response to shear stress, i.e., forces on the blood vessels in the direction of blood flow, and to mediators of inflammation. Furchgott and van Houtte (1989); Ignarro (1989).
In the immune system, the macrophage isoform is produced by activated macrophages and neutrophils as a cytotoxic agent. Nitric oxide produced in these cells targets tumor cells and pathogens. Hibbs et al. (1988); Nathan (1992); and Marletta (1989).
In the nervous system, the neuronal NOS isoform is localized to discrete populations of neurons in the cerebellum, olfactory bulb, hippocampus, cortex, striatum, basal forebrain, and brain stem: Bredt et al. (1990). NOS is also concentrated in the posterior pituitary gland, in the superoptic and paraventricular hypothalamic nuclei, and in discrete ganglion cells of the adrenal medulla Id. The widespread cellular localization of neuronal NOS and the short half-life and diffusion properties of nitric oxide suggest that it plays a role in nervous system morphogenesis and synaptic plasticity.
During development, NO may influence activity-dependent synaptic pruning, apoptosis, and the establishment of the columnar organization of the cortex. Gaily et al. (1990), Edelman and Gally (1992). Two forms of long-term synaptic modulation, long-term depression of the cerebellum, Shibuki and Okada (1991), and long-term potentiation (LTP) in the hippocampus, are sensitive to inhibitors of NOS. Bohme et al. (1991); Haley et al. (1992); O""Dell et al. (1991); Schuman and Madison (1991). Thus, nitric oxide may serve as a retrograde neurotransmitter to enhance synaptic function due to correlated firing of pre-and postsynaptic cells.
In the peripheral nervous system, nitric oxide mediates relaxation of smooth muscle. Smooth muscle relaxation in the gut, important to adaptation to a bolus of food and peristalsis, depends upon inhibitory non adrenergic, noncholinergic nerves that mediate their effects via nitric oxide. Boeckvstaens et al. (1991); Bult et al. (1990); Desai et al. (1991); Gillespie et al. (1989); Gibson et al. (1990); Ramagopal and Leighton (1989); Tottrup et al. (1991). NOS-containing neurons also innervate the corpus carvornosa of the penis, Burnett et al. (1992); Rajfer et al. (1992), and the adventitial layer of cerebral blood vessels. Nozaki et al. (1993); Toda and Okamura (1990). Stimulation of these nerves can lead to penile erection and dilation of cerebral arteries, respectively. These effects are blocked by inhibition of NOS.
Various biological roles of NO are described by Schmidt and Walter (1994); Nathan and Xie (1994); and Snyder (1995). The major roles of nitric oxide include:
(1) vasodilation or vasoconstriction with resulting change in blood pressure and blood flow;
(2) neurotransmission in the central and peripheral nervous system, including mediation of signals for normal gastrointestinal motility; and
(3) defense against pathogens like bacteria, fungus, and parasites due to the toxicity of high levels of NO to pathogenic organisms.
Recently, a role for NO has been proposed in the pathophysiology of cerebral ischemia, one form of vascular endothelial disorder. Iadecola et al. (1994); Dalkara and Moskowitz (1994). Since NO is diffuisible, short-lived, and reactive free radical gas that is difficult to measure in vivo, Archer (1993), most studies examining ischemic outcomes have based their conclusions on results following NOS inhibition by arginine analogues such as nitro-L-arginine or nitro-L-arginine methyl ester. These inhibitors, however, lack enzyme selectivity and block multiple isoforms. Rees et al. (1990). This nonselectivity might account in part for the discrepant outcomes after administration of NOS inhibitors following middle cerebral artery (MCA) occlusion.
Atherosclerosis is another form of a vascular endothelial disorder. This disease, a major cause of mobidity and mortality, is progressive beginning many years before the onset of overt symptoms. During the development of atherosclerosis, biochemical, cellular, and hemodynamic forces drive change in blood vessel walls, which ultimately leads to endothelial disfunction, cellular proliferation, recruitment of endothelial cells, and accumulation of oxidized LDL. Ross (1995). The cellular and molecular mechanisms that underlie these processes are complex. Rodent models offer the ability to study the contribution of individual genes, alone or in combination, to the molecular events in atherosclerosis. Breslow (1996); Ross (1996).
Cells within atherosclerotic plaques are monoclonal or oligoclonal in origin, indicating that intimal proliferation plays an important role in the development of lesions. Benditt (1973). Intimal proliferation also occurs as a common response to arterial injury of many kinds, regardless of whether the injury is luminal or advertitial. Schwartz (1995). Thus, niodels of vessel injury are relevant to atherosclerosis. For example, in a cuff model of adventitial injury, Booth et al. (1989); Kockx et al (1993), signals from the adventitia stimulate formation of a neointimal layer in a predictable manner. This model leaves the endothelium intact, so that the role of endothelial gene products can be studied. In a filament model of endothelial injury, Lindner (1993), the endothelium is physically removed, resulting in proliferation of medial smooth muscle cells. The rate at which endothelial cells resurface the injured areas can be quantitated.
In another type of atherosclerosis model, defined genetic mutations are used to increase the propensity of mice to atherosclerosis. Breslow (1996); Ross (1996). For example, mutant mice that form atherosclerotic lesions include apoE gene knockout mice, Plumb (1992); Zhang (1992); apoE Leiden mutation, van der Maagdenberg (1993); LDL receptor gene knockout mice, Ishibayashi (1993); and transgenic mice expressing the human apoB gene, Purcell-Huynh (1995). Of these, apoE knockout mice are an attractive atherosclerosis model, since they develop lesions on a low cholesterol, low-fat diet, and do not require the addition of cholic acid. These knockout mice develop fatty streaks that progress to fibrous plaques at branchpoints of major vessels, similar to human lesions. The rate and extent of lesion formation and its pathological severity can be quantitated. For example, a Western diet results in faster progression of the disease and formation of larger plaques than a low-cholesterol diet. Thus, the apoE knockout mice exhibit many aspects of human atherosclerosis.
Nitric oxide has physiological effects in blood vessels that may prevent atherosclerosis, such as suppression of smooth muscle proliferation, Mooradian (1995), inhibition of platelet aggregation and adhesion, Radomski (1991), and inhibition of leukocyte activation and adhesion, Bath (1993); Lefer (1993). It has also been recently reported that arginine inhibits atherosclerosis in LDL receptor mutant mice. Aji (1997).
While certain physiological effects of nitric oxide may prevent atherosclerosis, other studies suggest that excessive nitric oxide production may contribute to the development of atherosclerosis. Busse (1976); Leitinger (1995); Radomski (1995). Evidence for a pro-atherogenic role for nitric oxide includes several different findings. First, expression of iNOS and nNOS isoforms can be induced in atherosclerotic vessels. Aji (1997); Sobey (1995); Topors (1995); Wilcox (1994). Second, human atherosclerotic lesions contain nitrotyrosine, suggesting that peroxynitrite is formed in atherosclerotic lesions. See Beckman (1994b). Peroxynitrite is formed by the reaction of nitric oxide with superoxide in biological systems, Beckman (1994a), and is an extremely potent oxidant that can initiate lipid peroxidation of human LDL. Darley-Usmar (1992). Third, nitric oxide affects redox-sensitive transcription of genes involved in endothelial cell activation such as VCAM-1. This implicates nitric oxide in atherosclerosis.
However, prior to this invention, it was not clear which NOS isoforms were involved in stimulating atherosclerosis. Malinski (1993). Moreover, it was also not clear how important is peroxynitrite formation to the molecular events of atherosclerosis. Peroxynitrite (ONOOxe2x88x92) is a strong oxidant capable of lipid and protein oxidation. Beckman (1994a). Superoxide reacts with nitric oxide to form peroxynitrite faster, rate constant of 6.7xc3x9710xe2x88x929 M/sec, than superoxide is scavanged by superoxide dismutase, rate constant of 2.0xc3x9710xe2x88x929 M/sec. Endothelial cells are sensitive to the redox state and may respond with a program of endothelial cell activation, including expression of VCAM-1, ICAM-1, E-selectin, and MCP-1.
Many studies depend on pharmacological agents that block NOS enzymes, such as L-nitroarginine (L-NA) and L-N-arginine methyl ester (L-NAME). Inhibition of a process by these NOS inhibitors, and reversal of inhibition by excess L-arginine, but not D-arginine, provides evidence for the involvement of NO. However, these NOS inhibitors affect all three isoforms, so that the effect on different isoforms cannot be distinguished. Distinguishing between various NOS isoforms is particularly important since NOS isoforms have multiple roles and divergent effects.
Targeted gene disruption of the endothelial or neuronal NOS isoforms offers a new approach to dissect the relevance of NO in brain ischemia and the development of treatments for brain ischemia and stroke. For example, mice deficient in neuronal NOS gene expression were relatively resistant to brain injury after permanent focal cerebral ischemia. Huang et al. (1994).
Over the last several years, transgenic animals have been made containing specific genetic defects, e.g., resulting in the development of, or predisposition to, various disease states. These transgenic animals can be useful in characterizing the effect of such a defect on the organism as a whole, and developing pharmacological treatments for these defects.
The relevant techniques whereby foreign DNA sequences can be introduced into the mammalian germ line have been developed in mice. See Manipulating the Mouse Embryo (Hogan et al., eds., 2d ed., Cold Spring Harbor Press, 1994) (ISBN 0-87969-384-3). At present, one route of introducing foreign DNA into a germ line entails the direct microinjection of a few hundred linear DNA molecules into a pronucleus of a fertilized one-cell egg. Microinjected eggs may then subsequently be transferred into the oviducts of pseudo-pregnant foster mothers and allowed to develop. It has been reported by Brinster et al. (1985), that about 25% of the mice that develop inherit one or more copies of the micro-injected DNA.
In addition to transgenic mice, other transgenic animals have been made. For example, transgenic domestic livestock have also been made, such as pigs, sheep, and cattle.
Once integrated into the germ line, the foreign DNA may be expressed in the tissue of choice at high levels to produce a functional protein. The resulting animal exhibits the desired phenotypic property resulting from the production of the functional protein.
In light of the various biological functions of nitric oxide, there exists a need in the art to develop transgenic animals, e.g., transgenic mice, wherein the endothelial nitric oxide synthase gene has been modified. There also exists a need in the art to develop methods to test compounds for activity against various pathological states associated with the absence of eNOS, such as hypertension, atherosclerosis, and stroke, using these transgenic animals. A further need in the art is to develop treatments for various pathological states using nitric oxide or nitric oxide prodrugs.
This invention satisfies these needs in the art by providing a transgenic non-human animal comprising a disrupted endothelial nitric oxide synthase gene. In embodiments of this invention, the transgenic non-human animal exhibits hypertension or wound-healing abnormalities. In a specific embodiment of the invention, the transgenic non-human animal is a mouse. Moreover, in specific embodiments of this invention, the endothelial nitric oxide synthase gene is disrupted at exons encoding the NADPH ribose and adenine binding sites.
In other embodiments, this invention provides a method of testing compounds for activity against a vascular endothelial disorder by providing a transgenic non-human animal having a disrupted endothelial nitric oxide synthase gene, wherein the animal exhibits either a vascular endothelial disorder or is at increased risk of developing the vascular endothelial disorder, administering a compound to be tested to the transgenic animal, and determining the effect of the compound on the vascular endothelial disorder in the animal. In specific embodiments of this invention, the vascular endothelial disorder is hypertension or atherosclerosis.
This invention also provides a method for treating vascular endothelial disorders comprising: selectively increasing the expression of eNOS in blood vessels without increasing the expression of iNOS or nNOS. In specific embodiments, gene therapy approaches are used.
In a further embodiment, this invention provides a method of testing compounds for wound-healing activity by providing a transgenic non-human animal having a disrupted endothelial nitric oxide synthase gene, administering a compound to be tested to the transgenic animal, and determining the effect of the compound on the wound-healing capabilities of the animal.
In another embodiment, this invention provides a method of treating wounds by applying nitric oxide or a prodrug thereof. In a specific embodiment thereof, compounds which release nitric oxide into wounds to improve or speed up wound healing.
In an additional embodiment, this invention provides a method of making a transgenic non-human animal of the invention comprising providing an embryonic stem cell comprising an intact eNOS gene; providing a targeting vector capable of disrupting said eNOS gene upon homologous recombination; introducing said targeting vector into said cells under conditions where the intact eNOS gene of said cell and said targeting vector undergo homologous recombination to produce a disrupted eNOS gene; introducing said cells into a blastocyst; implanting the blastocyst into the uterus of a pseudopregnant female; and delivering transgenic animals of the invention from said pseudopregnant female. In order to obtain homozygous mutant mice of the invention, the resulting animals can be bred, and homozygous mutant mice selected.
In another embodiment, this invention provides a method of testing compounds for utility in the treatment of cerebral ischemia or stroke by providing a transgenic non-human animal having a disrupted endothelial nitric oxide synthase gene, administering a compound to be tested to the transgenic animal, determining the effect of the compound on infarct size or regional cerebral blood flow (rCBF) following induction of focal cerebral ischemia in the brain of said animal, and correlating the effect of said compound on infarct size or rCBF with a utility for the treatment of cerebral ischemia or stroke. In a preferred embodiment, the eNOS mutant animal has been rendered normotensive.
In a further embodiment, the invention also provides a method for testing compounds for utility in the treatment of atherosclerosis by providing a transgenic non-human animal having a disrupted endothelial nitric oxide synthase gene, administering a compound to be tested for atherosclerosis activity to the transgenic animal, determining the effect of the compound on atherosclerosis, and correlating the effect of said compound on atherosclerosis with a utility for the treatment of atherosclerosis. In specific embodiments, the effect of the compound on atherosclerosis is determined using the vessel or the cuff vessel injury model.